We identified four novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs), GRL-078, -079, -077, and -058, containing an alkylamine at the C5 position of P2-tetrahydropyrano-tetrahydrofuran (Tp-THF) and a P2' -cyclopropyl (Cp)(or isopropyl)-aminobenzothiazole (Abt). Their 50% effective concentrations (EC50s) were 2.5-30 nM against wild-type HIV-1NL4-3, 0.3-6.7 nM against HIV-2EHO, and 0.9-90 nM against laboratory-selected-PI-resistant HIV-1 and clinical HIV-1 variants resistant to multiple FDA-approved PIs (HIVMDR). GRL-078, -079, -077, and -058 also effectively blocked the replication of HIV-1 variants highly resistant to darunavir (DRV:HIVDRVRp51), with EC50 values of 38, 62, 61, and 90 nM, respectively, while four FDA-approved PIs examined [amprenavir, atazanavir, lopinavir (LPV), and DRV] virtually had no activity (EC50>1,000 nM) against HIVDRVRp51 Structurally, GRL-078, -079, and -058 form strong hydrogen bond interactions between the C5-modified-Tp-THF and Asp29/Asp30/Gly48 of wild-type protease while P2' -Cp-Abt forms strong hydrogen bonds with Asp30' . The Tp-THF and Cp-Abt moieties also have good nonpolar interactions with protease residues located in the flap region. When selected with LPV and DRV using a mixture of 11 HIVMDR strains (HIV11MIX), HIV11MIX became highly resistant to LPV and DRV over 13-32 and 32-41 weeks, respectively. However, when selected with GRL-079 and GRL-058, HIV11MIX failed to replicate at >0.08 μM and >0.2 μM, respectively. Thermal stability results supported the highly favorable anti-HIV-1 potency of GRL-079 as well as other PIs. The present data strongly suggest that the C5-modified P2-Tp-THF and P2' -Abt moiety contribute to the potent anti-HIV-1 profiles of the four PIs against HIV-1NL4-3 and a wide spectrum of HIVMDRs.

The HIV-2 protease (PR2) is a homodimer of 99 residues with asymmetric assembly and binding various ligands. We propose an exhaustive study of the local structural asymmetry between the two monomers of all available PR2 structures complexed with various inhibitors using a structural alphabet approach. On average, PR2 exhibits asymmetry in 31% of its positions-i.e., exhibiting different backbone local conformations in the two monomers. This asymmetry was observed all along its structure, particularly in the elbow and flap regions. We first differentiated structural asymmetry conserved in most PR2 structures from the one specific to some PR2. Then, we explored the origin of the detected asymmetry in PR2. We localized asymmetry that could be induced by PR2's flexibility, allowing transition from the semi-open to closed conformations and the asymmetry potentially induced by ligand binding. This latter could be important for the PR2's adaptation to diverse ligands. Our results highlighted some differences between asymmetry of PR2 bound to darunavir and amprenavir that could explain their differences of affinity. This knowledge is critical for a better description of PR2's recognition and adaptation to various ligands and for a better understanding of the resistance of PR2 to most PR2 inhibitors, a major antiretroviral class.

A recent advancement in isolation and cryopreservation has resulted in commercially available primary human enterocytes that express various drug metabolizing enzymes (DMEs) and transporters. The main objective of this study was to further evaluate the utility of pooled cryopreserved enterocytes, specifically MetMax™ cryopreserved human enterocytes (In Vitro ADMET Laboratories), as an in vitro model for assessing intestinal clearance in comparison to hepatocytes. It was found that, for CYP3A4 substrates such as midazolam, amprenavir and loperamide, in vitro metabolic clearance is generally lower in enterocytes compared to that of hepatocytes, which is consistent with the relative abundance of the enzyme between the intestine and liver. In contrast, raloxifene, a surrogate UGT activity substrate, showed 3-fold greater turnover in enterocytes than hepatocytes, which is likely attributed to the differential expression of individual UGTs in human liver and intestine. For procaine, a known CES2 substrate, the measured apparent clearance was higher in hepatocytes, but formation of 4-aminobenzoic acid, a CE2-specific metabolite, was more pronounced in enterocytes, suggesting that CE2 is more active in enterocytes. Salbutamol, a SULT1A3 substrate, showed little turnover in both enterocytes and hepatocytes, and more abundant sulfate conjugate was detected in enterocytes, indicating higher SULT activity in enterocytes than hepatocytes. As expected, ketoconazole inhibited CYP3A-mediated metabolite formation in enterocytes for midazolam, amprenavir and loperamide, suggesting that cryopreserved enterocytes may be useful in determining intestinal CYP3A inhibition parameters. Interestingly, elacridar, a P-gp inhibitor, suppressed metabolite formation in enterocytes for loperamide, a substrate of CYP3A4 and P-gp, suggesting that MetMax™ enterocytes do not have active P-gp efflux functions, and the suppression of metabolism in enterocytes is probably caused by inhibition of CYP3A4 by elacridar. Our results suggested that pooled cryopreserved human enterocytes, specifically the MetMax™ cryopreserved human enterocytes, represent a valuable in vitro model for assessing first-pass clearance and potential drug interactions in human intestine.

The use of highly active antiretroviral therapy has resulted in changes of comorbidity profile in people living with HIV (PLHIV), increasing non-AIDS-related events. The occurrence of cardiovascular events is greater in PLHIV, but the mechanism responsible for it is still controversial. This article aimed to investigate factors associated with the progression to cardiovascular events in PLHIV using HAART. A 15-years cohort study with 1135 PLHIV was conducted in Rio de Janeiro-Brazil. Clinical progression was stratified in five states: No comorbidities (s1), arterial hypertension (s2), lipid abnormalities (s3), hypertension and lipid abnormalities (s4) and major cardiovascular events (stroke, coronary artery disease, thrombosis or death) (s5). Semi-Markov models evaluated the effects of cardiovascular traditional factors, treatment and clinical covariates on transitions between these states. Hazard Ratios (HR) and 95% confidence intervals (CI) were provided. In addition to traditional factors (age, sex, educational level and skin color), the development of one comorbidity (lipid abnormalities or hypertension) increased in patients with low nadir CD4 (<50 cells/mm3), (HR = 1.59, CI 1.11-2.28 and 1.36, CI 1.11-1.66, respectively). The risk to experience a second comorbidity (s3→s4) increased 75% with low nadir CD4. Age was the only factor that increased the risk of major cardiovascular events once having lipid abnormalities with or without hypertension (s3,s4→s5). The prolonged use of certain antiretroviral drugs (abacavir, didanosine, ritonavir, lopinavir, amprenavir and fosamprenavir) increased the risk of direct transition (s1→s5) to major cardiovascular events (HR = 5.29, CI 1.16-24.05). This analysis suggests that prolonged use of certain antiretroviral drugs led directly to major cardiovascular events, while low nadir CD4 only affected the occurrence of lipid abnormalities and hypertension. Management strategies, including rational use of complex exams (such as, computed-tomography angiography), statins and antihypertensives, should be developed based on the distinct roles of antiretroviral use and of HIV infection itself on the progression to cardiovascular events.

A paucity of information is available on the activity of protease inhibitors (PI) in chronically-infected monocyte-derived macrophages (MDM) and on the kinetics of viral-rebound after PI removal in vitro. To fill this gap, the activity of different concentrations of amprenavir (AMP) was evaluated in chronically-infected MDM by measuring p24-production every day up to 12 days after drug administration and up to seven days after drug removal. Clinically-relevant concentrations of AMP (4 and 20 μM) drastically decreased p24 amount released from chronically-infected MDM from Day 2 up to Day 12 after drug administration. The kinetics of viral-rebound after AMP-removal (4 and 20 μM) showed that, despite an initial increase, p24-production over time never reached the level observed for untreated-MDM, suggesting a persistent intracellular drug activity. In line with this, after AMP-removal, human immunodeficiency virus 1 (HIV-1) infectivity and intracellular the p24/p55 ratio (reflecting virion-maturation) were remarkably lower than observed for untreated MDM. Overall, AMP shows high efficacy in blocking HIV-1 replication in chronically-infected MDM, persisting even after drug-removal. This highlights the role of protease inhibitors in preventing the establishment of this important HIV-1 reservoir, thus reducing viral-dissemination in different anatomical compartments.

Interactions with the human breast cancer resistance protein (hBCRP) significantly influence the pharmacokinetic properties of a drug and can even lead to drug-drug interactions. As efflux pump from the ABC superfamily, hBCRP utilized energy gained by adenosine 5'-triphosphate (ATP) hydrolysis for the transmembrane movement of its substrates, while adenosine 5'-diphosphate (ADP) and inorganic phosphate were released. The ADP liberation can be used to detect interactions with the hBCRP ATPase. An ADP quantification method based on hydrophilic interaction liquid chromatography (HILIC) coupled to high resolution tandem mass spectrometry (HR-MS/MS) was developed and successfully validated in accordance to the criteria of the guideline on bioanalytical method validation by the European Medicines Agency. ATP and adenosine 5'-monophosphate were qualitatively included to prevent interferences. Furthermore, a setup consisting of six sample sets was evolved that allowed detection of hBCRP substrate or inhibitor properties of the test compound. The hBCRP substrate sulfasalazine and the hBCRP inhibitor orthovanadate were used as controls. To prove the applicability of the procedure, the effect of amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir on the hBCRP ATPase activity was tested. Nelfinavir, ritonavir, and saquinavir were identified as hBCRP ATPase inhibitors and none of the five HIV protease inhibitors turned out to be an hBCRP substrate. These findings were in line with a pervious publication.

We report that GRL-09510, a novel HIV-1 protease inhibitor (PI) containing a newly-generated P2-crown-tetrahydrofuranylurethane (Crwn-THF), a P2'-methoxybenzene, and a sulfonamide isostere, is highly active against laboratory and primary clinical HIV-1 isolates (EC50: 0.0014-0.0028 μM) with minimal cytotoxicity (CC50: 39.0 μM). Similarly, GRL-09510 efficiently blocked the replication of HIV-1NL4-3 variants, which were capable of propagating at high-concentrations of atazanavir, lopinavir, and amprenavir (APV). GRL-09510 was also potent against multi-drug-resistant clinical HIV-1 variants and HIV-2ROD. Under the selection condition, where HIV-1NL4-3 rapidly acquired significant resistance to APV, an integrase inhibitor raltegravir, and a GRL-09510 congener (GRL-09610), no variants highly resistant against GRL-09510 emerged over long-term in vitro passage of the virus. Crystallographic analysis demonstrated that the Crwn-THF moiety of GRL-09510 forms strong hydrogen-bond-interactions with HIV-1 protease (PR) active-site amino acids and is bulkier with a larger contact surface, making greater van der Waals contacts with PR than the bis-THF moiety of darunavir. The present data demonstrate that GRL-09510 has favorable features for treating patients infected with wild-type and/or multi-drug-resistant HIV-1 variants, that the newly generated P2-Crwn-THF moiety confers highly desirable anti-HIV-1 potency. The use of the novel Crwn-THF moiety sheds lights in the design of novel PIs.

Although antiretroviral (ARV) therapy has reduced the incidence of severe dementia associated with HIV infection, there has been a rise in milder neurocognitive complaints. Data from HIV patients taking ARVs have shown measurable neurocognitive improvements during drug cessation, suggesting a neurotoxic role of the therapy itself. Mechanisms underlying potential ARV neurotoxicity have not been thoroughly investigated, however pathologic oxidative stress and mitochondrial dysfunction have been suspected. Using DIV 16 primary rat cortical neuron culture, we tested eight ARVs from the three most commonly prescribed ARV classes: nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs) for effects on neuron viability and morphology after 24 h of drug exposure. Of the tested NRTIs, only stavudine at nearly 100 times the target plasma concentration affected neuron viability with no appreciable change in morphology. Dideoxyinosine induced dendritic simplification at 100 times target plasma concentrations, but did not adversely affect viability. The sole NtRTI, tenofovir, induced dendritic simplification at approximately 3-4 times target plasma concentration, but did not affect viability. Of the tested PIs, only amprenavir decreased neuron viability at nearly 100 times the target plasma concentration. The non-nucleoside reverse transcriptase inhibitor, efavirenz, consistently reduced viability (at 50 µM) and induced dendritic simplification (at 20 µM) nearest the target plasma concentration. Probing mitochondrial energetics of DIV16 cortical neurons after exposure to 20 µM efavirenz showed rapid diminution of mitochondrial-dependent oxygen consumption. Further, 20 µM efavirenz decreased excitability in ex vivo slice culture whereas 2 µM had no effect.

The efficacy of HIV-1 protease (PR) inhibition therapies is often compromised by the emergence of mutations in the PR molecule that reduces the binding affinity of inhibitors while maintaining viable catalytic activity and affinity for natural substrates. In the present study, we used a recombinant HIV-1 C-SA PR and a recently reported variant for inhibition (Ki, IC50) and thermodynamic studies against nine clinically used inhibitors. This is the first time that binding free energies for C-SA PR and the mutant are reported. This variant PR harbours a mutation and insertion (I36T↑T) at position 36 of the C-SA HIV-1 PR, and did not show a significant difference in the catalytic effect of the HIV-1 PR. However, the nine clinically approved HIV PR drugs used in this study demonstrated weaker inhibition and lower binding affinities toward the variant when compared to the wild type HIV-1 PR. All the protease inhibitors (PIs), except Amprenavir and Ritonavir exhibited a significant decrease in binding affinity (p<0.0001). Darunavir and Nelfinavir exhibited the weakest binding affinity, 155- and 95-fold decreases respectively, toward the variant. Vitality values for the variant PR, against the seven selected PIs, confirm the impact of the mutation and insertion on the South African HIV-1 subtype C PR. This information has important clinical implications for thousands of patients in Sub-Saharan Africa.